Molecular cloning and sequencing of equine interleukin 4

Veterinary Immunology and Immunopathology, 40 (1994) 379-384 0165-2427/94/$07.00 © 1994 - Elsevier Science B.V. All fights reserved

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Short Communication Molecular cloning and sequencing of equine interleukin 4 Evie V. Vandergrifft, Cyprianna E. Swiderski, David W. Horohov* Department of VeterinaryMicrobiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70808, USA (Accepted 6 May 1993) Abstract

We have cloned equine interleukin4 ( IL-4 ) eDNA using the polymerase chain reaction (PCR) and primers based on the human 1I.,-4sequence. The eDNA was amplified from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC). The cloned PCR product shares extensive homology with IL-4 sequences from other species. Abbreviations

IL-4, interleukin 4; PBMC, peripheral blood mononuclear ceils; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PWM, pokeweed mitogen.

Introduction

Interleukin 4 (IL-4) is a pleiotropic T-cell-derived cytokine that acts on a variety of cells (Paul and Ohara, 1987; Yokota et al., 1988; Finkelman et al., 1990). IL-4 was originally described as a B-cell growth factor (Howard et al., 1982 ), inducing B-cell proliferation and immunoglobulin production, particularly IgG~ and IgE (Pene et at., 1988). In addition to B-cells, IL-4 acts on mast cells (Mosmann et al., 1986), monoeytes (Wieser et al., 1989), hemopoietic progenitor cells (Peschel et al., 1987; Rennick et al., 1987 ); and Tcells (Golumbek et al., 1991 ). IL-4 is highly mitogenic for activated T-cells and induces specific eytotoxic CD8 + cell activity (Widmer and Grabstein, 1987). The mouse, human and rat IL-4 eDNAs have been cloned and sequenced. IL-4 eDNA was first isolated from a mouse eDNA library using direct expression in COS cells and assaying the cell supernate for B-cell stimulatory activity (Lee et al., 1986 ). Once the mouse IL-4 had been cloned and sequenced, it was used as a probe to identify human IL-4 eDNA from a library (Yokota et al., 1986 ), and rat IL-4 eDNA was amplified using polymerase chain reac*Corresponding author. Tel.: 504-346-3362. Fax: 504-346-5715.

SSDIO165-2427(93)O5212-W

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tion (PCR) and primers based on the mouse IL-4 sequence (McKnight et al., 1991). The nucleotide sequence similarity allowed the use of primers specific for the human IL-4 sequence to amplify the coding region of equine IL-4 eDNA from pokeweed mitogen (PWM) stimulated PBMC. Materials and methods

Equine peripheral blood mononuclear cells (PBMC) were isolated from venous blood by differential centrifugation over Ficoll-Paque (Pharmacia LKB Biotechnology, Piscataway, N J). After washing in calcium chloride and magnesium free phosphate-buffered saline (PBS-CMF), the PBMC were suspended at a concentration of 10 6 m l - ~in RPMI 1640 (Sigma Chemical Co., St. Louis, MO ) supplemented with 2-mercaptoethanol ( 10- 8 M ), glutamine (2 raM), 100 U ml -~ penicillin, 100 gg ml -~ streptomycin and 5% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT ). PBMC were incubated with PWM (2 #g ml-1; Sigma) at 39°C in a humidified environment with 5% CO2.

Total RNA isolation and complementary DNA synthesis Total RNA was isolated at 24 h intervals from the PBMC cultures. Smallscale RNA preparations, using 2-3 × 106 cells, were performed as outlined by Brenner et al. (1989), using the Cytokine MAPPing Protocol (Message Amplification Phenotyping, Clonetech Laboratories, Palo Alto, CA). Complementary DNA (eDNA) was synthesized using Moloney Murine Leukemia Virus reverse transcriptase and an oligo d-T primer (Gibco-BRL, Gaithersburg, M D ) (Sambrook et al., 1989).

Equine IL-4 message amplification The PCR reaction was performed using 4 pmol of the human IL-4 amplimers, 5'-ATGGGTCTCACCTCCCAACTGCT-3' and 5'-CGAACACTTTGAATATTTCTCTCTCAT-3' (Clonetech Laboratories). Degenerative conditions were used, specifically 35 cycles with denaturation at 96 °C for 30 s, a low annealing temperature of 40°C for 30 s and extension at 72 °C for 60 s. To facilitate sequencing and further DNA manipulations, the 396 bp PCR fragment was cloned into the SmaI site of vector pUC 19 (Yanish-Perron et al., 1985).

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Sequencing of equine IL-4 cDNA Equine IL-4 eDNA was sequenced by chain-termination sequencing of the double-stranded pUC19 derivative by alkali denaturation according to the Sequencase Version 2.0 kit (US Biochemical, Cleveland, OH). Two unique clones were sequenced using appropriate overlapping sequence runs and combination of primers; the -40 primer (US Biochemical), -48 primer (GeneLab, Baton Rouge, LA), the h u m a n cytokine amplimers (Clonetech) and one internal primer that was based on a 3' consensus region within the h u m a n and mouse IL-4. The sequence of the internal primer was 5' G A T C G T C T T T A G C C T T T C C A A G A A G 3 ' (GeneLab). The sequence information was entered into the MacVector database program for subsequent alignment with published eDNA for h u m a n IL-4. Results

Equine IL-4 cDNA PCR time course PCR amplification of eDNA from 48, 72 and 96 h PWM-activated samples produced a distinct 396 bp fragment (Fig. 1 ). Amplification of eDNA from unstimulated cells yielded no PCR products (data not shown). As a single

1

2

3

4

5

6

7

,2 8}

Fig. 1. PCR amplification of equine IL-4. Lanes 1 and 7 contain the size marker phage X 174 DNA HaeIIIdigested (the size of the fragments in base pairs is given); lane 2 is the positive human IL-4 PCR control; lanes 3-6 contain the PCR products from PWM stimulated equine PBMC eDNA for Days 1-4, respectively.

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defined PCR product was obtained only from mitogen-simulated samples, it was purified and cloned for sequencing.

Sequencing and amino acid analysis of equine IL-4 cDNA The nucleotide sequence of the cDNA insert is shown in Fig. 2 and the aligned deduced amino acid sequence is given in Fig. 3. The cDNA insert contained a single open reading frame of 132 amino acids starting with an initiator methionine codon ATG. The PCR amplified cDNA did not contain a termination codon. The precursor equine IL-4 protein has a predicted molecular weight of 15 283. Comparison with other known secretory proteins (Watson, 1984), and with the crystallized structure of human IL-4 (Powers et al., 1992), suggests that a signal sequence is cleaved off during secretion, making phenylalanine the amino terminal residue. The mature equine IL-4 5 '-

ATGGGTCTCA TACCAGCTTC TCAAAACGCT CTGACTGTAG CTGCAGGGCT TGATCAAAGA GGGACCTGCT GGAAAGGCTA

CCTCCCAACT ATCCAGGGAT GAACCTCACA CGGATGCCTT GCAAAGGTGC ATGCCTGAGC GTACTGTGAA AAGACGATCA

GCTTCCAGCT GCAAATACGA GATGGAAAGG TGGCCCGAAG TTCAACAGTA GGACTGGACA TGAAGCCAAG TGAGAGAGAA

CTGGTCTGCT CATCACCTTA GCAAGAATTC AACACAGATG TAAAAGACAT GGAACAAGGG AAGAGCACAT ATATTCAAAG

TACTAGCATG CAAGAGATCA GTGCATGGAG GAAAGGAAAT GACAGGTCCT CATGGCAAAC TGGACTTTTT T G T T C G - 3'

50 I00 150 200 250 300 350

Fig. 2. Nucleotide sequence of amplified equine IL-4 cDNA. The nucleotide sequence of the cDNA insert is given as determined by chain termination sequencing of double-stranded plasmid DNA. This nucleotide sequence has been submitted to EMBL/GenBank, under accession no. L06010. 7

EQUINE HUMAN RAT MURINE

MGLTSQLLPA

FIQG..CKYD

MGLTSQLLPP LFFLLACAGN I~WItG..HKCD MGLSPHLAVT MGLNPQLVVI

28 NSCMELTVAD • .CTELTVTD • .C T E M F V P D • .C T E M D V P N

LVCLLACTSN LFCFLICTGN LIFFLECTRS

GIHG .... CN HIHG .... CD

50 AFAGPKNTDG IFAASKNTTE VLTATRNTTE VLTATKNTTE

LNNLTDGKGK

.ITIA~EIIKT I.~SLTEQKTL LNQVTEKGTP LNEVTGEGTP

69

KE.ICRAAKV KETFCRAATV NELICRASRV SELVCRASKV

LQQLYKRHDR LRQFYSHHEK LRKFYFPRDV LRIFYLKHGK

SLIKECL ............. DTR..CLGAT AQQFHRHKQL •P P . . C L K N K S G V L G E L R K L .TP. . C L K K N S S V L M E L Q R L

TVNEAKKST. PVKEANQST. TVNEST.LTT TMNESK.STS

LKDFLERL~T LENFLERLKT LKDFLESLKS LKDFLESLKS

IMREKYSK.C IMREKYSK.C ILRGKYLQSC IMQMD~S

10B .... S G L D R N L K G M A N G T C C IRFLKRLDRN LWGLAGLNSC CRGVSGLNS ....... LRSC FRAFRCLDS ....... SISC

.ITLQEIIKT DSPLREIINT KNNLREIIGI

131 S SS TSMS

Fig. 3. Deduced amino acid sequence of equine IL-4 aligned with peptide sequences of human, rat and murine IL-4. Putative signal peptides are in italics. Cysteine residues believed to be involved in intramolecular bonding are designated with an asterisk. Numbering of amino acids is with reference to the mature human amino acid sequence• Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Ash; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

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would have a relative mass of 12 815. Predicted molecular weight does not take into consideration any post-translational glycosylation of the polypeptide. Computer analysis of amino acid sequences for equine, human, mouse and rat IL-4 demonstrated extensive similarity. Equine IL-4 amino acid sequence shares 62% similarity with the human IL-4 and 40% similarity with the mouse and rat IL-4 amino acid sequences. Discussion Using the polymerase chain reaction with primers specific for the human IL-4 sequence, we have amplified an equine cDNA that shows considerable homology to IL-4 sequences of other species. This is the first report of cloning equine IL-4. Equine IL-4 contains seven cysteine residues in the proposed mature peptide. The six cysteines that are involved in disulfide bonds in the human IL-4 are conserved in equine and rat IL-4, suggesting similar intramolecular disulfide binding. The amplified eDNA did not contain a stop codon. Although the serine at position 132 may represent the carboxy terminal residue, it is possible that additional amino acids may be present in the native equine IL-4. It has been reported that the carboxy terminus of the molecule is crucial for biological function (Le et al., 1991 ). A critical residue in the carboxy terminus of mature human IL-4 is cys TM, which is involved in a disulfide bond with cys7 (Powers et al., 1992 ); a similar bond may occur between equine residues cys 5 and cys TM. AS the critical carboxy cysteine is present in equine IL-4, additional amino acids at the carboxy terminus may not be necessary for biological activity. This will be determined by expression of this cDNA.

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